Insights into secondary structure of nucleic acids have yielded powerful tools in mutation detection and organism identification. Several analytical techniques have been developed that take advantage of different secondary structures that exist between one sample and the next. Some of these techniques include Single-Stranded Conformational Polymorphisms (SSCP) (Orita et al., PNAS (USA) 86:2766–2770 (1989)), heteroduplex mobility assay (HMA) (Espejo et al., Microbiology 144:1611–1617 (1998)), ribotyping (Grimont and Grimont, Ann. Inst. Pasteur Microbiol. 137B(2):165–175 (1989)) and post-PCR product identification by product melting temperature (Tm) (Ririe et al., Anal. Biochem. 245154–160 (1997)).
Secondary structure analysis methods focus on detecting nucleic acid characteristics that depend on how specific sequences interact with each other. In 1989, Orita et al. demonstrated that a single base pair deletion or alteration is detectable by analyzing single-stranded conformational polymorphisms evident in nucleic acid gel electrophoresis (Orita, et al. 1989). Essentially, she found that small differences in the primary nucleic acid sequence result in slightly different secondary structure conformations, which migrate differentially in an electrophoresis gel matrix. SSCP analysis is very sensitive and even more specific than restriction fragment length polymorphism (RFLP) methods (Orita, et al 1989).
Similar to SSCP are Heteroduplex Mobility Assays or HMA. In HMA, the electrophoretic mobility of a non-perfectly matched DNA target-probe duplex is monitored (Espejo et al., 1998). The mismatching within the target-probe duplex causes secondary structure distortions resulting in mobility variations of the duplex. Differences in target sequences are therefore made evident by differential product migration in an electrophoresis gel matrix (Espejo et al., 1998).
Ribotyping is the identification of ribosomal nucleic acid gene restriction patterns observed by gel electrophoresis (Grimont and Grimont, 1989). Ribotyping takes advantage of the ubiquitous nature of ribosomes, the hypervariable regions found within their nucleic acid components and the specificity of restriction enzymes In this method, genes coding for rRNA, containing hypervariable regions flanked by conserved regions, are amplified using PCR and the DNA produced is subsequently enzymatically digested. Specific restriction enzymes are selected for restriction sites within the hypervariable regions. DNA cleavage by these enzymes results in small segments of DNA of varying lengths. Following enzymatic cleavage the DNA fragments are run on a gel and analyzed. A particular combination (pattern) of DNA segments is therefore indicative of the primary sequence. And if enzymes are chosen properly, each organism type will have a unique combination of restriction fragments (Grimont and Grimont, 1989; Van Camp, Curr. Microbiol. 27(3):147–151 (1993)).
Sequencing of ribosomal nucleic acids (rRNA) has identified many hypervariable regions surrounded by highly conserved regions (De Rijk et al., Nuc. Acids Res. 20 (Supplement):2075–2089 (1992); Edwards et al., Nuc. Acids Res., 17(19):7843–7853 (1989); Grimont and Grimont, 1989; Van Camp, 1993). Indeed, many universal primer sites, capable of amplifying DNA from a wide variety of organisms, have been identified and adopted for SSCP or ribotyping analysis (Van Camp, 1993; Weisburg et al., J. Bacteriol. 173 (2):697–703 (1991); Stubbs et al., J. Clin. Microbiol. 37(2):461–463 (1999); Anthony et al., J. Clin. Microbiol. 38(2):781–788 (2000); Rantakokko-Jalava et al., J. Clin. Microbiol. 38(1):32–39 (2000); Widjojoatmodjo et al., J. Clin. Microbiol. 32(12):3002–3007 (1994)). Work performed by these research groups has found medically significant regions within the 16s and 23s genes that contain secondary structures within hypervariable segments. Van Camp and colleagues identified several universal primer sites which can be used to amplify hypervariable regions within the 23s gene (1993). The work done by Widjojoatmodjo demonstrates good species typing by SSCP with a small amplicon size, ranging from 108 bp to 300 bp (1994). And, Erik Avaniss-Aghajani et al. identified and tested a primer set “capable of amplifying the SSU [small subunit] rRNA from essentially all bacteria” for bacterial typing using ribotyping techniques (Biotechiques. 17(1):144–146, 148–149 (1994)).
In each of the analytical methods described above (SSCP, HMA and ribotyping), nucleic acid sequences are identified by secondary structural analysis. All three of these processes are highly sensitive to sequence variations and can be used to identify differences in nucleic acid sequences that exist between organisms. However, the assays all depend on gel electrophoresis, and ribotyping requires the additional step of enzymatic cleavage. Both gel electrophoresis and enzymatic cleavage are time-consuming post-amplification steps.
In another form of secondary structural analysis of nucleic acids, primary sequence variations are made evident by observing double stranded nucleic acid melting characteristics. Melting of nucleic acids refers to the conformational transition from a doublehelical state to a single-stranded state. The temperature at which half of the nucleic acid strands are in the doublehelical state and half are in the ‘random coil’ (single stranded) state is defined as the melting temperature (Tm). (Santa Lucia, PNAS (USA) 95:1460–1465 (1998)). The Tm of a given pair of nucleic acid strands therefore, is indicative of the stability of the strand to strand binding and depends on the strands' complementarity, sequence length, GC content and environmental conditions (Lewin, Genes V, Chapter 5, Oxford University Press and Cell Press: New York, (1994) pp. 109–126; SantaLucia, 1998).
The analysis of nucleic acid melting has been accomplished in many ways. Methods to observe and analyze nucleic acid denaturation transitions include: measuring the enthalpy change within a sample as it denatures by differential scanning calorimetry (DSC) (Kulinski et al., Nucleic Acids Res. 19(9):2449–2455 (1991); Paner et al., Biopolymers 29:1715–1734 (1990); Volker et al., Biopolymers 50:303–318 (1999)), measuring the fluorescence of covalently attached pairs of fluorophores (Vamosi and Clegg, Biochemistry 37:14300–14316 (1998)), and monitoring the change in hyperchromicity of nucleic acids (Haugland, “In Vitro Applications for Nucleic Acid Stains and Probes”, in Handbook of Fluorescent Probes and Research Chemicals, 6th ed., Molecular Probes Inc, Eugene OR (1996) pp. 161–174). DSC is a technique which was first used to measure the purity of a chemical. The process measures the heat evolved or absorbed during chemical reactions or transitions (Plato, Anal. Chem. 41(2):330–336 (1969)). Detailed analysis and development of theoretical models of nucleic acid transitions have been possible using DSC techniques (Paner et al., 1990). Kulinski observed different melting profiles of two plant 5S rRNA segments obtained from Lupin seeds and Wheat germ using DSC (Kulinski, 1991). Unfortunately, both optical (hyperchromicity) and DSC analyses requires a substantial quantity of nucleic acid, analysis is slow and usually only single samples can be studied at a time. And, the measure of fluorescence resonance energy transfer between paired fluorophors, as described in Vamosi and Clegg (1998), requires the covalent attachment of the fluorophors at termini of duplexed nucleic acid, which termini must be adjacent when the nucleic acid is duplexed for effective measurement.
Tm values of double stranded nucleic acids can also be observed by monitoring the fluorescence of double-stranded DNA-specific dyes combined with the nucleic acids (Wittwer et al., 1996). Double stranded-specific dyes are nucleic acid-binding fluorophors. Typically, the fluorescence of these dyes increases when bound to duplexed nucleic acids (Wittwer et al., BioTechniques 22:176–181 (1997)). Ririe et al. (1997) demonstrated that post PCR products can be differentiated by melting curve analysis using the double stranded nucleic acid binding dye SYBR® Green I. SYBR® Green I binds preferentially to double stranded nucleic acid (Haugland, 1996).
The process of Tm analysis does not require additional post-PCR handling. However, current applications using double stranded DNA-specific dyes, such as SYBR Green I, are not sequence specific and have not been used to differentiate organisms with one primer set. Furthermore, these dyes have only been used in the analysis of separate, complementary strands of nucleic acid.
In light of the foregoing discussion, it is apparent that there is a need for faster and simpler methods of analysis of single stranded nucleic acid. Such methods should be applicable to both identifying variations in a sequence, such as SSCP and HMA, and typing organisms, such as ribotyping.